目的:研究Synaptotagm inⅡ(简称Syt2)在RBL-2H3(简称RBL)中的表达及其在胞吐中的作用。方法:通过W estern b lot检测Syt2在RBL中的表达情况。用高保真酶扩增Syt2,与pEGFP-N1构建全长反义基因表达载体,电穿孔转染RBL,G418筛选获得稳定转染细胞。通过钙离子载体和抗原刺激,应用W estern b lot检测稳定转染细胞和对照细胞分泌的组织蛋白酶D,分析Syt2对胞吐的影响。结果:在RBL中检测到Syt2表达。构建了pEGFP-N1-Syt2-AS质粒,插入片段测序结果与GenBank登录号NM012665(rat Syt2)序列完全一致。经转染和G418筛选,获得了稳定转染细胞RBL-Syt2-AS,W estern b lot结果显示,两株RBL-Syt2-AS表达的Syt2均明显减少,分别只有对照的8%和10%。经刺激后,RBL-Syt2-AS分泌的组织蛋白酶D较对照明显增加。结论:Syt在RBL中表达,其对RBL溶酶体胞吐起负调控作用。
Summary: The expression of synaptotagmin Ⅱ(Syt2) in RBL-2H3 (RBL) and its role during exocytosis of RBL was investigated. The expression of Syt2 in RBL was detected by western blot and Syt2 gene was amplified by PCR. The anti-sense full length Syt2 cDNA expression vector was constructed with pEGFP-N1 and transfected into RBL by electroporation, and stable transfectants were selected by using G418. To analyze the role of Syt2 during exocytosis of RBL, the release of cathepsin D was assayed by immunoblotting. The results showed that Syt2 was expressed in RBL. The anti-sense expression vector pEGFP-N1-Syt2-AS was constructed and the sequence of insertion was completely consistent with rat Syt2 (accession number in GeneBank : NM012665). The stable transfectants (RBL-Syt2-AS) were obtained. Western blot showed that RBL-Syt2-AS expressed a lower level of Syt2 (8 % and 10 % of control cells), indicating that the expression of Syt2 in RBL-Syt2-AS was markedly down-regulated by anti-RNA. Compared with control, the release of cathepsin D by RBL-Syt2-AS was increased. It was concluded that Syt2 expressed in RBL and could inhibit exocytosis of lysosomes in RBL.
